This invention relates to novel pharmaceutically useful compounds, in particular competitive inhibitors of trypsin-like serine proteases, especially thrombin, their use as medicaments, pharmaceutical compositions containing them and synthetic routes to their production.
Blood coagulation is the key process involved in both haemostasis (i.e. the prevention of blood loss from a damaged vessel) and thrombosis (i.e. the formation of a blood clot in a blood vessel, sometimes leading to vessel obstruction).
Coagulation is the result of a complex series of enzymatic reactions. One of the ultimate steps in this series of reactions is the conversion of the proenzyme prothrombin to the active enzyme thrombin.
Thrombin is known to play a central role in coagulation. It activates platelets, leading to platelet aggregation, converts fibrinogen into fibrin monomers, which polymerise spontaneously into fibrin polymers, and activates factor XIII, which in turn crosslinks the polymers to form insoluble fibrin. Furthermore, thrombin activates factor V and factor VIII leading to a xe2x80x9cpositive feedbackxe2x80x9d generation of thrombin from prothrombin.
By inhibiting the aggregation of platelets and the formation and crosslinking of fibrin, effective inhibitors of thrombin would be expected to exhibit antithrombotic activity. In addition, antithrombotic activity would be expected to be enhanced by effective inhibition of the positive feedback mechanism.
The early development of low molecular weight inhibitors of thrombin has been described by Claesson in Blood Coagul. Fibrinol. (1994) 5, 411.
Blombxc3xa4ck et al (in J. Clin. Lab. Invest. 24, suppl. 107, 59, (1969)) reported thrombin inhibitors based on the amino acid sequence situated around the cleavage site for the fibrinogen Axcex1 chain. Of the amino acid sequences discussed, these authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1, hereinafter referred to as the P3-P2-P1 sequence) would be the most effective inhibitor.
Thrombin inhibitors based on dipeptidyl derivatives with an xcex1,xcfx89-aminoalkyl guanidine in the P1-position are known from U.S. Pat. No. 4,346,078 and International Patent Application WO 93/11152. Similar, structurally related, dipeptidyl derivatives have also been reported. For example International Patent Application WO 94/29336 discloses compounds with, for example, aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic aminoalkyl guanidines in the P1-position; European Patent Application 0 648 780, discloses compounds with, for example, cyclic aminoalkyl guanidines in the P1-position.
Thrombin inhibitors based on peptidyl derivatives, also having cyclic aminoalkyl guanidines (e.g. either 3- or 4-aminomethyl-1-amidinopiperidine) in the P1-position are known from European Patent Applications 0 468 231, 0 559 046 and 0 641 779.
Thrombin inhibitors based on tripeptidyl derivatives with arginine aldehyde in the P1-position were first disclosed in European Patent Application 0 185 390.
More recently, arginine aldehyde-based peptidyl derivatives, modified in the P3-position, have been reported. For example, International Patent Application WO 93/18060 discloses hydroxy acids, European Patent Application 0 526 877 des-amino acids, and European Patent Application 0 542 525 O-methyl mandelic acids in the P3-position.
Inhibitors of serine proteases (e.g. thrombin) based on electrophilic ketones in the P1-position are also known. For example, European Patent Application 0 195 212 discloses peptidyl xcex1-keto esters and amides, European Patent Application 0 362 002 fluoroalkylamide ketones, European Patent Application 0 364 344 xcex1,xcex2,xcex4-triketocompounds, and European Patent Application 0 530 167 xcex1-alkoxy ketone derivatives of arginine in the P1-position.
Other, structurally different, inhibitors of trypsin-like serine proteases based on C-terminal boronic acid derivatives of arginine and isothiouronium analogues thereof are known from European Patent Application 0 293 881.
More recently, thrombin inhibitors based on peptidyl derivatives have been disclosed in European Patent Application 0 669 317 and International Patent Applications WO 95/35309, WO 95/23609 and WO 96/25426.
However, there remains a need for effective inhibitors of trypsin-like serine proteases, such as thrombin. There is a particular need for compounds which are both orally bioavailable and selective in inhibiting thrombin over other serine proteases. Compounds which exhibit competitive inhibitory activity towards thrombin would be expected to be especially useful as anticoagulants and therefore in the therapeutic treatment of thrombosis and related disorders.
According to the invention there is provided a compound of formula I, 
wherein
R1 represents H, C1-4 alkyl (optionally substituted by one or more substituents selected from cyano, halo, OH, C(O)OR1a or C(O)N(R1b)R1c) or OR1d;
R1d represents H, C(O)R11, SiR12R13R14 or C1-6 alkyl, which latter group is optionally substituted or terminated by one or more substituent selected from OR15 or (CH2)qR16;
R12, R13 and R14 independently represent H, phenyl or C1-6 alkyl;
R16 represents C1-4 alkyl, phenyl, OH, C(O)OR17 or C(O)N(H)R18;
R18 represents H, C1-4 alkyl or CH2C(O)OR19;
R15 and R17 independently represent H, C1-6 alkyl or C1-3 alkylphenyl;
R1a, R1b, R1c, R11 and R19 independently represent H or C1-4 alkyl; and
q represents 0, 1 or 2;
Rx represents a structural fragment of formula IIa, IIb or IIc, 
wherein
the dotted lines independently represent optional bonds;
A and B independently represent O or S, CH or CH2 (as appropriate), or N or N(R21) (as appropriate);
D represents xe2x80x94CH2xe2x80x94, O, S, N(R22), xe2x80x94(CH2)2xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CH2N(R22)xe2x80x94, xe2x80x94N(R22)CH2xe2x80x94, xe2x80x94CHxe2x95x90Nxe2x80x94, xe2x80x94Nxe2x95x90CHxe2x80x94, xe2x80x94CH2Oxe2x80x94, xe2x80x94OCH2xe2x80x94, xe2x80x94CH2Sxe2x80x94 or xe2x80x94SCH2xe2x80x94;
X1 represents C2-4 alkylene; C2-3 alkylene interrupted by Z; xe2x80x94C(O)-Z-A1; -Z-C(O)-A1-; xe2x80x94CH2xe2x80x94(O)-A1; -Z-C(O)-Z-A2-; xe2x80x94CH2-Z-C(O)-A2-; -Z-CH2-C(O)-A2-; -Z-CH2-S(O)m-A2-; xe2x80x94CH2-Z-S(O)m-A2-; xe2x80x94C(O)-A3; -Z-A3-; or -A3-Z-;
X2 represents C2-3 alkylene, xe2x80x94C(O)-A4- or -A4-C(O)xe2x80x94;
X3 represents CH or N;
X4 represents a single bond, O, S, C(O), N(R23), xe2x80x94CH(R23)xe2x80x94, xe2x80x94CH(R23)xe2x80x94CH(R24)xe2x80x94 or xe2x80x94C(R23)xe2x95x90C(R24)xe2x80x94;
A1 represents a single bond or C1-2 alkylene;
A2 represents a single bond or xe2x80x94CH2xe2x80x94,
A3 represents C1-3 alkylene;
A4 represents C(O) or C1-2 alkylene;
Z represents, at each occurrence, O, S(O)m or N(R25);
m represents, at each occurrence, 0, 1 or 2;
R2 and R4 independently represent one or more optional substituents selected from C1-4 alkyl (which latter group is optionally substituted by one or more halo substituent), C1-4 alkoxy, methylenedioxy, halo, hydroxy, cyano, nitro, SO2NH2, C(O)OR26 or N(R27)R28);
R3 represents an optional substituent selected from OH or C1-4 alkoxy;
R21, R22, R23, R24, R25, R26, R27 and R28 independently represent H or C1-4 alkyl;
Y represents CH2, (CH2)2, CHxe2x95x90CH, (CH2)3, CH2CHxe2x95x90CH or CHxe2x95x90CHCH2, which latter three groups are optionally substituted by C1-4 alkyl, methylene, oxo or hydroxy;
Ry represents H or C1-4 alkyl;
n represents 0, 1, 2, 3 or 4; and
B represents a structural fragment of formula IIIa, IIIb or IIIc 
wherein
X5, X6, X7 and X8 independently represent CH, N or Nxe2x80x94O;
X9 and X10 independently represent a single bond or CH2; and
R31 represents an optional substituent selected from halo and C1-4 alkyl;
or a pharmaceutically acceptable salt thereof;
provided that:
(a) A and B do not both represent O or S;
(b) B and D do not both represent O or S;
(c) when R1 represents OR1d and X1 represents xe2x80x94C(O)-Z-A1, -Z-CH2-S(O)m-A2-, xe2x80x94CH2-Z-S(O)m-A2- or -Z-C(O)-Z-A2, then A1 or A2 (as appropriate) do not represent a single bond; and
(d) when X4 represents xe2x80x94CH(R23)xe2x80x94, R1 does not represent OH.
The compounds of formula I may exhibit tautomerism. All tautomeric forms and mixtures thereof are included within the scope of the invention. Further it will be appreciated by those skilled in the art that, in the structural fragment of formula IIa, the optional double bonds, may, in conjunction with certain identities of substituent D, render the ring bearing A, B and D aromatic in character.
The compounds of formula I may also contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism. All diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques. Alternatively the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation, or by derivatisation, for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means (e.g. HPLC, chromatography over silica). All stereoisomers are included within the scope of the invention.
Alkyl groups which R1, R1a, R1b, R1c, R1d, R2, R4, R11, R12, R13, R14, R15, R16, R17, R18, R19, R21, R22, R23, R24, R25, R26, R27, R28, R31 and Ry may represent, and with which Y may be substituted; the alkyl part of alkylphenyl groups which R15 and R17 may represent; and alkoxy groups which R2, R3 and R4 may represent, may, when there is a sufficient number of carbon atoms, be linear or branched, saturated or unsaturated, cyclic or acyclic. Alkylene groups which X1, X2, A1, A3 and A4 may represent may, when there is a sufficient number of carbon atoms, be linear or branched, saturated or unsaturated.
Halo groups, which R31 may represent, and with which R1, R2 and R4 may be substituted, include fluoro, chloro, bromo and iodo.
In the structural fragments of formulae IIa, IIb and IIc, the dots indicate the carbon atom which is bonded to the xe2x80x94C(O)xe2x80x94 group and to R1 in a compound of formula I (for the avoidance of doubt, there is no further H atom bonded to the carbon atom so indicated).
The wavy lines on the bond in the fragments of formulae IIIa, IIIb and IIIc signify the bond position of the fragment.
According to a further aspect of the invention there is provided a compound of formula I as hereinbefore defined with the additional provisos that:
Ry represents H;
R28 represents H;
X4 does not represent xe2x80x94CH(R23)xe2x80x94.
According to a further aspect of the invention there is provided a compound of formula I as hereinbefore defined with the additional provisos that:
Ry represents C1-4 alkyl;
R28 represents C1-4 alkyl;
X4 represents xe2x80x94CH(R23)xe2x80x94.
Abbreviations are listed at the end of this specification.
When n represents 2 and B represents a structural fragment of formula IIIb, preferred compounds of formula I include those wherein X9 and X10 do not both represent CH2.
Preferred compounds of formula I include those wherein:
R1 represents OH or C1-4 alkyl (which latter group is optionally substituted by cyano or OH);
Rx represents a structural fragment of formula IIa;
when Rx represents a structural fragment of formula IIa, the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94;
when Rx represents a structural fragment of formula IIa, X1 represents C2- or C3-alkylene, xe2x80x94OCH2xe2x80x94 or xe2x80x94O(CH2)2xe2x80x94;
Y represents CH2, (CH2)2 or (CH2)3;
B represents a structural fragment of formula IIIa in which X5, X6, X7 and X8 all represents CH.
More preferred compounds of the invention include those wherein, when Rx represents a structural fragment of formula IIa, X1 represents C3-alkylene or xe2x80x94O(CH2)2xe2x80x94.
When Rx represents a structural fragment of formula IIa, and R2 represents at least one substituent, a preferred point of substitution is at the carbon atom which is at position B.
When Rx represents a structural fragment of formula IIa, the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94 (i.e. the ring bearing R2 is a benzo group), and R2 represents at least one substituent, the ring is preferably substituted either at the carbon atom in the xe2x80x94CHxe2x95x90CHxe2x80x94 group (position D) which is adjacent to the ring junction, or, more preferably, at the carbon atom which is at position B, or at both of these sites. For example, when the fragment IIa represents a tetralin-1-yl group (i.e. the dotted lines represent bonds, A and B both represent CH, D represents xe2x80x94CHxe2x95x90CHxe2x80x94 and X1 represents saturated C3-alkylene), preferred substitution positions are at the 5- or, especially, at the 7-position, or at both to of these positions. Correspondingly, when the fragment IIa represents a chroman-4-yl group (i.e. the dotted lines represent bonds, A and B both represent CH, D represents xe2x80x94CHxe2x95x90CHxe2x80x94 and X1 represents xe2x80x94O(CH2)2xe2x80x94), preferred substitution positions are at the 8- or, especially, at the 6-position, or at both of these positions.
Compounds of formula I in which the fragment 
is in the S-configuration are preferred. The wavy lines on the bonds in the above fragment signify the bond position of the fragment.
Preferred compounds of formula I include the compounds of the Examples described hereinafter.
According to the invention there is also provided a process for the preparation of compounds of formula I which comprises:
(i) the coupling of a compound of formula IV, 
wherein R1 and Rx are as hereinbefore defined with a compound of formula V, 
wherein Ry, Y, n and B are as hereinbefore defined; or
(ii) the coupling of a compound of formula VI, 
wherein R1, Rx, and Y are as hereinbefore defined with a compound of formula VII,
H(Ry)Nxe2x80x94(CH2)nxe2x80x94Bxe2x80x83xe2x80x83VII 
wherein Ry, n and B are as hereinbefore defined,
for example in the presence of a coupling agent (e.g. oxalyl chloride in DMF, EDC, DCC, HBTU, HATU or TBTU), an appropriate base (e.g. pyridine, 2,4,6,-trimethylpyridine, DMAP, TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane, acetonitrile or DMF).
Compounds of formula IV are commercially available, are well known in the literature, or are available using known and/or standard techniques.
For example, compounds of formula IV in which R1 represents OH may be prepared by reaction of a compound of formula VIII,
Rxxe2x95x90Oxe2x80x83xe2x80x83VIII 
wherein Rx is as hereinbefore defined, with:
(a) KCN, for example at 20xc2x0 C. in the presence of sodium bisulphite in water, followed by hydrolysis in the presence of aqueous acid (e.g. HCl), for example at 20xc2x0 C. in the presence of a suitable solvent (e.g. alcohol and/or water);
(b) CHCl3, in the presence of aqueous base (e.g. NaOH);
(c) TMSCN, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. CH2Cl2), followed by hydrolysis in the presence of acid (e.g. HCl or H2SO4), for example at 20xc2x0 C. (e.g. according, or analogously, to the method described by Bigge et al in J. Med. Chem. (1993) 36, 1977), followed by alkaline hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents H may be prepared from corresponding compounds of formula IV in which R1 represents OH (or a lower alkyl ester of the acid), for example by elimination of water, followed by hydrogenation of the resultant alkene using techniques which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents C1-4 alkyl may be prepared from corresponding compounds of formula IV in which R1 represents H (or a lower alkyl ester of the acid), for example by reaction with an appropriate alkyl halide using techniques which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula IV in which R1 represents OR1d and R1d represents C(O)R11, SiR12R13R14 or C1-6 alkyl may be prepared by acylation, silylation or alkylation (as appropriate) of a corresponding compound of formula IV in which R1 represents OH (or a lower alkyl ester of the acid) under conditions which are well known to those skilled in the art, followed by, if necessary, hydrolysis to give the free acid.
Compounds of formula V may be prepared by reaction of a compound of formula IX 
wherein Y is as hereinbefore defined with a compound of formula VII as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I.
Compounds of formulae V and VII in which Ry represents C1-4 alkyl may be prepared by reaction of a corresponding compound of formula V or formula VII, as appropriate, in which Ry represents H with a compound of formula IXa,
RyHalxe2x80x83xe2x80x83IXa 
wherein Hal represents halo (e.g. Cl, Br or I) and Ry is as hereinbefore defined, for example under conditions which are well known to those skilled in the art.
Compounds of formula VI are readily available using known techniques. For example, compounds of formula VI may be prepared by reaction of a compound of formula IV as hereinbefore defined with a compound of formula IX as hereinbefore defined, for example under conditions such as those described hereinbefore for synthesis of compounds of formula I.
Compounds of formula VIII are commercially available, are well known in the literature, or may be prepared in accordance with known techniques. For example compounds of formula VIII may be prepared as follows:
(a) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents C2-4 alkylene, xe2x80x94Z-A3- or xe2x80x94C(O)-A3-, in which A3 is as hereinbefore defined; and R3 is absent, may be prepared by cyclisation of a compound of formula X, 
wherein X1a represents C2-4 alkylene, xe2x80x94Z-A3- or xe2x80x94C(O)-A3-, and Z, A3 and R2 are as hereinbefore defined, using an appropriate acylating agent. for example at 100xc2x0 C. in the presence of polyphosphoric acid or using PCl5 at reflux followed by AlCl3. Compounds of formula X in which X1a represents C3-alkylene or xe2x80x94C(O)-A3-, in which A3 represents C2-alkylene, may be prepared in accordance with known techniques, for example by reaction of succinic anhydride with the corresponding phenyl lithium and, for compounds of formula X in which X1a represents C3-alkylene, selective reduction of the resultant ketone, under conditions which are well known to those skilled in the art. Compounds of formula X in which X1a represents xe2x80x94Z-A3- and A3 represents C2-3 alkylene may be prepared as described hereinafter.
(b) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents C2-4 alkylene or xe2x80x94C(O)-A3-, in which A3 is as hereinbefore defined; and R3 is absent, may alternatively be prepared by cyclisation of a compound of formula XI, 
wherein R represents C1-6 alkyl and X1a and R2 are as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. an alkali metal alkoxide) and an appropriate organic solvent (e.g. lower alkyl alcohol) followed by hydrolysis and decarboxylation. Compounds of formula XI may be prepared in accordance with known techniques. For example, compounds of formula XI in which X1a represents C3-alkylene or xe2x80x94C(O)-A3- in which A3 represents C2-alkylene may be prepared by reaction of succinic anhydride with a compound of formula XII, 
wherein R1 represents C1-6 alkyl and R and R2 are as hereinbefore defined and, for compounds of formula XI in which X1a represents C3-alkylene, selective reduction of the resultant ketone, followed by functional group transformations of the amide and the acid to ester groups, under conditions which are well known to those skilled in the art.
(c) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94Z-A3- in which A3 represents C2 alkylene and Z represents O or S; and R3 is absent, may be prepared by cyclisation of a compound of formula XIII, 
wherein Hal and R2 are as hereinbefore defined, for example at 20xc2x0 C. in the presence of aqueous-ethanolic NaOH. For corresponding compounds of formula VIII in which X1 represents xe2x80x94Z-A3- and Z represents S(O)m in which m is 1 or 2, this abovementioned cyclisation should be followed by carrying out an oxidation reaction on the cyclised product comprising an S atom, for example using m-chloroperbenzoic acid.
(d) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94Z-A3- in which A3 represents C2-alkylene or xe2x80x94Zxe2x80x94C(O)-A1 in which A1 represents C1-alkylene; and R3 is absent, may be prepared by reaction of a compound of formula XIV, 
wherein R2 and Z are as hereinbefore defined, with either:
(1) for compounds of formula VIII in which X1 represents xe2x80x94Z-A3- in which A3 represents C2-alkylene, a compound of formula XV,
H2Cxe2x95x90CHxe2x80x94CO2Rxe2x80x83xe2x80x83XV 
wherein R is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. triethylamine or sodium ethoxide) and an appropriate organic solvent (e.g. ethanol or DMF); or
(2) a compound of formula XVI,
L1-G-CH2xe2x80x94CO2Rxe2x80x83xe2x80x83XVI 
wherein L1 represents a suitable leaving group (such as Cl, Br, I, mesylate or tosylate), G represents CH2 or C(O) and R is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. triethylamine) and an appropriate organic solvent (e.g. THF); followed by cyclisation under appropriate conditions (e.g. those described hereinbefore).
(e) Compounds of formula VIII in which R represents a structural fragment of formula IIa, in which the ring bearing A, B and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; X1 represents xe2x80x94CH2xe2x80x94Zxe2x80x94C1-2 alkylene-, in which Z is as hereinbefore defined; and R3 is absent, may be prepared by reaction of a compound of formula XVII, 
xe2x80x83
wherein the ring bearing Aa, Ba and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I, and Z and R2 are as hereinbefore defined, with a compound of formula XVIII,
L1-Alk-CO2Hxe2x80x83xe2x80x83XVIII 
wherein Alk represents C1-2 alkylene and L1 is as hereinbefore defined, for example at 20xc2x0 C. in the presence of a suitable base (e.g. sodium methoxide) and an appropriate organic solvent (e.g. THF).
(f) Compounds of formula VIII in which Rx represents a structural fragment of formulae IIb, IIc or IIa, in which latter case the ring bearing A, B and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; and, in the cases when Rx represents a structural fragment of formulae IIa or IIb, R3 is absent, may be prepared by cyclisation of a compound of formula XX,
Rxaxe2x80x94CO2Hxe2x80x83xe2x80x83XX 
wherein Rxa represents a structural fragment of formula XXa, XXb or XXc 
wherein, in XXa, the ring bearing Aa, Ba and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I, and R2, R4, X1, X2, X3 and X4 are as hereinbefore defined, in the presence of polyphosphoric acid, for example at 100xc2x0 C. The dots adjacent to the carbon atoms in fragments of formula XXa, XXb and XXc signify the point of attachment of the fragments to the CO2H group of the compound of formula XX. Compounds of formula XX may be prepared by hydrolysis of a corresponding compound of formula XXI,
Rxaxe2x80x94CO2Rxe2x80x83xe2x80x83XXI 
wherein Rax and R are as hereinbefore defined (and in which the CO2H in the fragments of formulae XXa, XXb and XXc in Rxa may also be replaced by CO2R), for example under reaction conditions which are well known to those skilled in the art.
(g) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa in which the ring bearing A, B and D is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I; X1 represents xe2x80x94Oxe2x80x94CH2xe2x80x94; and R3 is absent, may be prepared by reaction of a compound of formula XXII, 
wherein the ring bearing Aa, Ba and Da is a carbocyclic aromatic, or heterocyclic aromatic, ring as defined hereinbefore in respect of compounds of formula I, and R2, Hal and R are as hereinbefore defined, with diazomethane, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. diethyl ether).
(h) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94C(O)xe2x80x94Oxe2x80x94CH2xe2x80x94; and R3 is absent, may be prepared by cyclisation of a compound of formula XXIII, 
wherein R2 and R are as hereinbefore defined, for example at xe2x88x9220xc2x0 C. in the presence of sulphuric acid and an appropriate organic solvent (e.g. methanol). Compounds of formula XXIII may be prepared by reacting a corresponding acid halide with diazomethane, for example at 20xc2x0 C. in the presence of a suitable organic solvent (e.g. diethyl ether).
(i) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa or IIc in which X1 includes N(R25), or X4 represent N(R23), (as appropriate), and R23 and R25 (as appropriate) represent C1-4 alkyl may be prepared by reaction of a corresponding compound of formula VIII in which X1 includes, or X4 represents, (as appropriate) NH with a compound of formula XXV
Ra-Halxe2x80x83xe2x80x83XXV 
wherein Ra represents C1-4 alkyl and Hal is as hereinbefore defined, for example under conditions which are well known to those skilled in the art.
j) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa, in which the dotted lines represent bonds, A and B both represent CH and D represents xe2x80x94CHxe2x95x90CHxe2x80x94; X1 represents xe2x80x94C(O)xe2x80x94N(H)xe2x80x94CH2xe2x80x94; and R3 is absent, may be prepared by catalytic hydrogenation of an hydroxamic acid of formula XXVI, 
wherein R2 is as hereinbefore defined, using an appropriate catalyst system e.g. Pd/C) in the presence of a suitable organic solvent (e.g. methanol). Compounds of formula XXVI may be prepared by cyclisation of a corresponding compound of formula XXVII, 
wherein R2 is as hereinbefore defined, for example at 20xc2x0 C. in the presence of fuming HCl and tin dichloride.
(k) Selective oxidation of a compound of formula XXX,
Hxe2x80x94Rxxe2x80x94Hxe2x80x83xe2x80x83XXX 
wherein Rx is as hereinbefore defined, for example in the presence of a suitable oxidising agent (e.g. CrO3 or KMnO4) and an appropriate solvent (e.g. water).
(l) Selective oxidation of a compound of formula XXXI,
Hxe2x80x94Rxxe2x80x94OHxe2x80x83xe2x80x83XXXI 
wherein Rx is as hereinbefore defined, for example in the presence of a suitable oxidising agent (e.g. MnO2) in an appropriate organic solvent (e.g. CH2Cl2).
(m) Hydrolysis of an oxime formula XXXII,
Rxxe2x95x90Nxe2x80x94OHxe2x80x83xe2x80x83XXXII 
wherein Rx is as hereinbefore defined, for example by heating in the presence of acid (e.g. HCl) and an appropriate organic solvent. Compounds of formula XXXII may be prepared by reaction of a corresponding compound of formula XXX, as hereinbefore defined, with propyl nitrite, for example in the presence of HCl in ethanol.
(n) Compounds of formula VIII in which Rx represents a structural fragment of formula IIa and X1 represents xe2x80x94CH2xe2x80x94CHxe2x95x90CHxe2x80x94, may be prepared by elimination of a compound of formula XXXIII, 
wherein L2 represents a suitable leaving group (e.g. Br or SePh) and the dotted lines, A, B, D, R2 and R3 are as hereinbefore defined, under appropriate reaction conditions, for example in the presence of aqueous ethanolic NaOH or hydrogen peroxide, and an appropriate organic solvent (e.g. THF).
(o) Compounds of formula VIII in which Rx represents a structural fragment of formula IIb, X2 represents xe2x80x94C(O)-A4- and A4 is as hereinbefore defined, may be prepared by cyclisation of a compound of formula XXXIV, 
wherein Rb represents H, C1-6 alkyl or Hal and R2, R3, A4, X3 and Hal are as hereinbefore defined, for example in the presence of polyphosphoric acid, as described hereinbefore or, in the case where Rb represents Hal, in the presence of AlCl3 in nitromethane at, for example, 20xc2x0 C.
(p) Compounds of formula VIII in which Rx represents a structural fragment of formula IIb and X2 represents -A4-C(O)xe2x80x94 and A4 represents C1-2 alkylene may be prepared by cyclisation of a compound of formula XXXV, 
wherein A4a represents C1-2 alkylene and Hal, R2, R3 and X3 are as hereinbefore defined.
Compounds of formulae VII, IX, IXa, XII, XIII, XIV, XV, XVI, XVII, XVIII, XXI, XXII, XXV, XXVII, XXX, XXXI, XXIII, XXIV and XXV are either commercially available, are well known in the literature, or are available using known techniques, including techniques which are the same as, or analogous to, those described herein.
Substituents on the aromatic and/or non-aromatic, carbocyclic and/or heterocyclic ring(s) in compounds of formulae I, IV, V, VI, VII, VIII, X, XI, XII, XIII, XIV, XVII, XX, XXI, XXII, XXIII, XXVI, XXVII, XXX, XXXI, XXXII, XXXIII, XXXIV and XXV may be interconverted using techniques well known to those skilled in the art. For example, nitro may be reduced to amino, hydroxy may be alkylated to give alkoxy, alkoxy may be hydrolysed to hydroxy, alkenes may be hydrogenated to alkanes, halo may be hydrogenated to H, etc.
The compounds of formula I may be isolated from their reaction mixtures using conventional techniques.
It will be appreciated by those skilled in the art that in the process described above the functional groups of intermediate compounds may need to be protected by protecting groups.
Functional groups which it is desirable to protect include hydroxy, amino and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g. t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and tetrahydropyranyl. Suitable protecting groups for carboxylic acid include C1-6 alkyl or benzyl esters. Suitable protecting groups for amino, amidino and guanidino include t-butyloxycarbonyl, benzyloxycarbonyl or 2-trimethylsilylethoxycarbonyl (Teoc). Amidino and guanidino nitrogens may also be protected by hydroxy or alkoxy groups, and may be either mono- or diprotected.
The protection and deprotection of functional groups may take place before or after coupling, or before or after any other reaction in the abovementioned schemes.
In particular, the compounds of formula I may be prepared by processes comprising the coupling of an N-acylated amino acid or a N-protected amino acid. When a N-protected amino acid is used, the acyl group may be introduced after coupling. Deprotection of the nitrogen atom may then be effected using standard methods.
Protecting groups may be removed in accordance with techniques which are well known to those skilled in the art and as described hereinafter.
Certain protected derivatives (which may also be referred to as xe2x80x9cintermediatesxe2x80x9d) of compounds of formula I, which may be made prior to a final deprotection stage to form compounds of formula I, are novel.
According to a further aspect of the invention there is provided a compound of formula Ia, 
wherein B1 represents a structural fragment of formula IIId, IIIe or IIIf 
wherein D1 and D2 independently represent H, OH, ORa, OC(O)Rb, OC(O)ORc, C(O)ORd, C(O)Re; in which
Ra represents phenyl, benzyl, C1-7 alkyl (which latter group is optionally interrupted by oxygen or is optionally substituted by halo) or xe2x80x94C(Rf)(Rg)xe2x80x94OC(O)Rh;
Rb represents C1-17 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy, amino or halo); C1-6 alkoxy, C3-7 cycloalkyl, phenyl, naphthyl or C1-3 alkylphenyl (which latter five groups are optionally substituted by C1-6 alkyl or halo); or xe2x80x94[C(Ri)(Rj)]mOC(O)Rk;
Rc represents C1-17 alkyl, phenyl, 2-naphthyl (which latter three groups are optionally substituted by C1-6 alkyl, Si(Raa)(Rab)(Rac) or halo), xe2x80x94[C(Rm)(Rn)]nOC(O)Rp, or xe2x80x94CH2xe2x80x94Ar1;
Rd represents 2-naphthyl, phenyl, C1-3 alkylphenyl (which latter three groups are optionally substituted by C1-6 alkyl, C1-6 alkoxy, nitro, Si(Rba)(Rbb)(Rbc) or halo), C1-12 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy or halo), xe2x80x94[C(Rq)(Rr)]pOC(O)Rs or xe2x80x94CH2xe2x80x94Ar2;
Re represents phenyl, benzyl, C1-6 alkyl (which latter group is optionally interrupted by oxygen) or xe2x80x94[C(Rt)(Ru)]rOC(O)Rv;
Raa, Rab, Rac, Rba, Rbb and Rbc independently represent C1-6 alkyl or phenyl; Rf, Rg, Ri, Rj, Rm, Rn, Rq, Rr, Rt and Ru independently represent H or C1-6, alkyl;
Rh, Rk, Rp, Rs and Rv independently represent C1-17 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy or halo); C1-6 alkoxy, C3-7 cycloalkyl, phenyl, naphthyl or C1-3 alkylphenyl (which latter five groups are optionally substituted by C1-6 , alkyl or halo);
Ar1 and Ar2 independently represent the structural fragment 
m and r independently represent 3 or 4;
n and p independently represent 1, 2 or 3; and
R1, Rx, Y, Ry, n, X5, X6, X7, X8, X9, X10 and R31 are as hereinbefore defined;
or a pharmaceutically acceptable salt thereof;
provided that D1 and D2 do not both represent H.
Alkyl groups which Ra, Raa, Rab, Rac, Rb, Rba, Rbb, Rbc, Rc, Rd, Re, Rf, Rg, Rh, Ri, Rj, Rk, Rm, Rn, Rp, Rq, Rr, Rs, Rt, Ru and Rv may represent and with which Rb, Rc, Rd, Rh, Rk, Rp, Rs and Rv may be substituted; cycloalkyl groups which Rb, Rh, Rk, Rp, Rs and Rv may represent; the C1-3 alkyl part of alkylphenyl groups which Rb, Rd, Rh, Rk, Rp, Rs and Rv may represent; alkoxy groups which Rb, Rh, Rk, Rp, Rs and Rv may represent; and alkoxy and acyloxy groups with which Rb, Rd, Rh, Rk, Rp, Rs and Rv may be substituted, may, when there is a sufficient number of carbon atoms, be linear or branched, and may be saturated or unsaturated.
Halo groups with which Ra, Rb, Rc, Rd, Rh, Rk, Rp, Rs and Rv may be substituted include fluoro, chloro, bromo and iodo.
The wavy lines on the bond in the fragments of formulae IIId, IIIe or IIIf signify the bond position of the fragment.
Preferred compounds of formula Ia include those wherein D1 represents H and D2 represents OH, OCH3, OC(O)Rb or C(O)ORd, wherein Rb and Rd are as hereinbefore defined.
Compounds of formula Ia may also be prepared directly from compounds of formula I in accordance with techniques well known to those skilled in the art.
For example, compounds of formula Ia in which D1 or D2 represents C(O)ORd may be prepared by reaction of a corresponding compound of formula I with a compound of formula XXXVa,
L3-C(O)ORdxe2x80x83xe2x80x83XXXVa 
wherein L3 represents a leaving group such as Hal or p-nitrophenoxy, and Hal and Rd are as hereinbefore defined for example at 0xc2x0 C. in the presence of a suitable base (e.g. NaOH) and an appropriate organic solvent (e.g. THF).
Compounds of formula Ia may also be prepared directly from other compounds of formula Ia in accordance with techniques well known to those skilled in the art.
Compounds of formula Ia in which D1 or D2 represents OH may be prepared by reaction of a corresponding compound of formula Ia in which D1 or D2 (as appropriate) represents COORd and Rd is as hereinbefore defined with hydroxylamine (or a hydrohalide salt thereof), for example at 40xc2x0 C. in the presence of a suitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF).
Compounds of formula Ia in which D1 or D2 represents OC(O)ORc, and Rc is as hereinbefore defined, may be prepared by reaction of a corresponding compound of formula Ia in which D1 or D2 (as appropriate) represents OH with a compound of formula XXXVI,
L3C(O)ORcxe2x80x83xe2x80x83XXXVI 
wherein L3 and Rc are as hereinbefore defined, for example at room temperature in the presence of a suitable base (e.g. TEA, pyridine or DMAP) and an appropriate organic solvent.
Compounds of formula Ia in which D1 or D2 represents OC(O)Rb, and Rb is as hereinbefore defined, may be prepared by reaction of a corresponding compound of formula Ia in which D1 or D2 (as appropriate) represents OH with a compound of formula XXXVIa,
RbC(O)L4xe2x80x83xe2x80x83XXXVIa 
wherein L4 represents a suitable leaving group such as OH, Hal or RbC(O)O, and Hal and Rb are as hereinbefore defined, for example at or below room temperature in the presence of a suitable base (e.g. TEA, pyridine or DMAP) and an appropriate organic solvent (e.g. CH2Cl2).
Compounds of formula Ia in which B1 represents a structural fragment of formula IIId (in which X5, X6, X7 and X8 all represent CH) or IIIf, in which, in both cases, D1 represents H and D2 represents OH or ORa wherein Ra is as hereinbefore defined may alternatively be prepared by reaction of a compound of formula XXXVII, 
wherein Ba represents phenyl-1,4-ene or cyclohexyl-1,4-ene and R1, Rx, Y, Ry and n are as hereinbefore defined with a compound of formula XXXVIII,
H2NORa1tm XXXVIII 
wherein Ra1 represents H or Ra and Ra is as hereinbefore defined, for example at between 40 and 60xc2x0 C., in the presence of a suitable base (e.g. TEA) and an appropriate organic solvent (e.g. THF, CH3CN, DMF or DMSO). Compounds of formula Ia in which D1 or D2 represents OH or ORa may alternatively be prepared in an analogous fashion by reaction of a corresponding compound of formula Ia, wherein D1 or D2 (as appropriate) represent C(O)ORd, and Rd is as hereinbefore defined, with a compound of formula XXXVIII, as defined above.
Compounds of formula XXXVII may be prepared in accordance with peptide coupling techniques, for example in analogous fashion to the methods described hereinbefore for compounds of formula I. Compounds of formulae XXXVa, XXXVI, XXXVIa and XXXVIII are commercially available, are well known in the literature, or are available using known techniques.
According to a further aspect of the invention there is provided a compound of formula Ia as defined above except that:
Rb and Rc independently represent C1-17 alkyl, phenyl or 2-naphthyl (all of which are optionally substituted by C1-6 alkyl or halo);
Rd represents 2-naphthyl, phenyl, C1-3 alkylphenyl (which latter three groups are optionally substituted by C1-6 alkyl, C1-6 alkoxy, nitro or halogen), CH(Rf)(CH(Rg))pOC(O)Rh (in which Rf and Rg independently represent H or C1-6 alkyl, Rh represents 2-naphthyl, phenyl, C1-6 alkoxy or C1-8 alkyl (which latter group is optionally substituted by halo, C1-6 alkoxy or C1-6 acyloxy), and p represents 0 or 1) or C1-12 alkyl (which latter group is optionally substituted by C1-6 alkoxy, C1-6 acyloxy or halogen);
Ra and Re independently represent phenyl, benzyl, (CH2)2OC(O)CH3 or C1-6 alkyl which latter group is optionally interrupted by oxygen;
or a pharmaceutically acceptable salt thereof.
Persons skilled in the art will appreciate that, in order to obtain compounds of formula I, or formula Ia, in an alternative, and, on some occasions, more convenient, manner, the individual process steps mentioned hereinbefore may be performed in a different order, and/or the individual reactions may be performed at a different stage in the overall route (i.e. substituents may be added to and/or chemical transformations performed upon, different intermediates to those mentioned hereinbefore in conjunction with a particular reaction). This may negate, or render necessary, the need for protecting groups. Accordingly, the order and type of chemistry involved will dictate the need, and type, of protecting groups as well as the sequence for accomplishing the synthesis.
The use of protecting groups is fully described in xe2x80x98Protective Groups in Organic Chemistryxe2x80x99, edited by J W F McOmie, Plenum Press (1973), and xe2x80x98Protective Groups in Organic Synthesisxe2x80x99, 2nd edition, T W Greene and P G M Wutz, Wiley-Interscience (1991).
The protected derivatives of compounds of formula I (e.g. compounds of formula Ia) may be converted chemically to compounds of formula I using standard deprotection techniques (e.g. hydrogenation), for example as described hereinafter.
It will also be appreciated by those skilled in the art that, although such protected derivatives of compounds of formula I (e.g. compounds of formula Ia) may not possess pharmacological activity as such, they may be administered parenterally or orally and thereafter metabolised in the body to form compounds of formula I which are pharmacologically active. Such derivatives may therefore be described as xe2x80x9cprodrugsxe2x80x9d. All prodrugs of compounds of formula I are included within the scope of the invention.
Protected derivatives of compounds of formula I which are particularly useful as prodrugs include compounds of formula Ia.
Compounds of formula I, pharmaceutically-acceptable salts, tautomers and stereoisomers thereof, as well as prodrugs thereof (including compounds of formula Ia which are prodrugs of compounds of formula I), are hereinafter referred to together as xe2x80x9cthe compounds of the inventionxe2x80x9d.
The compounds of the invention are useful because they possess pharmacological activity. They are therefore indicated as pharmaceuticals.
According to a further aspect of the invention there is thus provided the compounds of the invention for use as pharmaceuticals.
In particular, the compounds of the invention are potent inhibitors of thrombin either as such or, in the case of prodrugs, after administration, for example as demonstrated in the tests described below.
The compounds of the invention are thus expected to be useful in those conditions where inhibition of thrombin is required.
The compounds of the invention are thus indicated in the treatment and/or prophylaxis of thrombosis and hypercoagulability in blood and tissues of animals including man.
It is known that hypercoagulability may lead to thrombo-embolic diseases. Conditions associated with hypercoagulability and thrombo-embolic diseases which may be mentioned include inherited or acquired activated protein C resistance, such as the factor V-mutation (factor V Leiden), and inherited or acquired deficiencies in antithrombin III, protein C, protein S, heparin cofactor II. Other conditions known to be associated with hypercoagulability and thrombo-embolic disease include circulating antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi, heparin induced thrombocytopenia and defects in fibrinolysis. The compounds of the invention are thus indicated both in the therapeutic and/or prophylactic treatment of these conditions.
The compounds of the invention are further indicated in the treatment of conditions where there is an undesirable excess of thrombin without signs of hypercoagulability, for example in neurodegenerative diseases such as Alzheimer""s disease.
Particular disease states which may be mentioned include the therapeutic and/or prophylactic treatment of venous thrombosis and pulmonary embolism, arterial thrombosis (eg in myocardial infarction, unstable angina, thrombosis-based stroke and peripheral arterial thrombosis) and systemic embolism usually from the atrium during arterial fibrillation or from the left ventricle after transmural myocardial infarction.
Moreover, the compounds of the invention are expected to have utility in prophylaxis of re-occlusion (ie thrombosis) after thrombolysis, percutaneous trans-luminal angioplasty (PTA) and coronary bypass operations; the prevention of re-thrombosis after microsurgery and vascular surgery in general.
Further indications include the therapeutic and/or prophylactic treatment of disseminated intravascular coagulation caused by bacteria, multiple trauma, intoxication or any other mechanism; anticoagulant treatment when blood is in contact with foreign surfaces in the body such as vascular grafts, vascular stents, vascular catheters, mechanical and biological prosthetic valves or any other medical device; and anticoagulant treatment when blood is in contact with medical devices outside the body such as during cardiovascular surgery using a heart-lung machine or in haemodialysis.
In addition to its effects on the coagulation process, thrombin is known to activate a large number of cells (such as neutrophils, fibroblasts, endothelial cells and smooth muscle cells). Therefore, the compounds of the invention may also be useful for the therapeutic and/or prophylactic treatment of idiopathic and adult respiratory distress syndrome, pulmonary fibrosis following treatment with radiation or chemotherapy, septic shock, septicemia, inflammatory responses, which include, but are not limited to, edema, acute or chronic atherosclerosis such as coronary arterial disease, cerebral arterial disease, peripheral arterial disease, reperfusion damage, and restenosis after percutaneous trans-luminal angioplasty (PTA).
Compounds of the invention that inhibit trypsin and/or thrombin may also be useful in the treatment of pancreatitis.
According to a further aspect of the present invention, there is provided a method of treatment of a condition where inhibition of thrombin is required which method comprises administration of a therapeutically effective amount of a compound of the invention, or a pharmaceutically acceptable salt thereof, to a person suffering from, or susceptible to such a condition.
The compounds of the invention will normally be administered orally, intravenously, subcutaneously, buccally, rectally, dermally, nasally, tracheally, bronchially, by any other parenteral route or via inhalation, in the form of pharmaceutical preparations comprising active compound either as a free base, or a pharmaceutical acceptable non-toxic organic or inorganic acid addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to be treated and the route of administration, the compositions may be administered at varying doses.
The compounds of the invention may also be combined and/or co-administered with any antithrombotic agent with a different mechanism of action, such as the antiplatelet agents acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane receptor and/or synthetase inhibitors, fibrinogen receptor antagonists, prostacyclin mimetics and phosphodiesterase inhibitors and ADP-receptor (P2T) antagonists.
The compounds of the invention may further be combined and/or co-administered with thrombolytics such as tissue plasminogen activator (natural, recombinant or modified), streptokinase, urokinase, prourokinase, anisoylated plasminogen-streptokinase activator complex (APSAC), animal salivary gland plasminogen activators, and the like, in the treatment of thrombotic diseases, in particular myocardial infarction.
According to a farther aspect of the invention there is thus provided a pharmaceutical formulation including a compound of the invention, in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier.
Suitable daily doses of the compounds of the invention in therapeutical treatment of humans are about 0.001-100 mg/kg body weight at peroral administration and 0.001-50 mg/kg body weight at parenteral administration.
The compounds of the invention have the advantage that they may be more efficacious, be less toxic, be longer acting, have a broader range of activity, be more potent, produce fewer side effects, be more easily absorbed than, or that they may have other useful pharmacological properties over, compounds known in the prior art.
Test A
Determination of Thrombin Clotting Time (TT)
The inhibitor solution (25 xcexcL) was incubated with plasma (25 xcexcL) for three minutes. Human thrombin (T 6769; Sigma Chem Co) in buffer solution, pH 7.4 (25 xcexcL) was then added and the clotting time measured in an automatic device (KC 10; Amelung).
The clotting time in seconds was plotted against the inhibitor concentration, and the IC50TT was determined by interpolation.
IC50TT is the concentration of inhibitor in the test that doubles the thrombin clotting time for human plasma.
Test B
Determinaton of Thrombin Inhibition with a Chromogenic, Robotic Assay
The thrombin inhibitor potency was measured with a chromogenic substrate method, in a Plato 3300 robotic microplate processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using 96-well, half volume microtitre plates (Costar, Cambridge, Mass., USA; Cat No 3690). Stock solutions of test substance in DMSO (72 xcexcL), 1 mmol/L, were diluted serially 1:3 (24+48 xcexcL) with DMSO to obtain ten different concentrations, which were analysed as samples in the assay. 2 xcexcL of test sample was diluted with 124 xcexcL assay buffer, 12 xcexcL of chromogenic substrate solution (S-2366, Chromogenix, Mxc3x6lndal, Sweden) in assay buffer and finally 12 xcexcL of (xcex1-thrombin solution, (Human xcex1-thrombin, Sigma Chemical Co.) both in assay buffer, were added, and the samples mixed. The final assay concentrations were: test substance 0.00068-13.3 xcexcmol/L, S-2366 0.30 mmol/L, xcex1-thrombin 0.020 NIHU/mL. The linear absorbance increment during 40 minutes incubation at 37xc2x0 C. was used for calculation of percentage inhibition for the test samples, as compared to blanks without inhibitor. The IC50-robotic value, corresponding to the inhibitor concentration which caused 50% inhibition of the thrombin activity, was calculated from a log dose vs. % inhibition curve.
Test C
Determinaton of the Inhibition Constant K1 for Human Thrombin
K1 determinations were made using a chromogenic substrate method, performed at 37xc2x0 C. on a Cobas Bio centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme activity after incubation of human xcex1-thrombin with various concentrations of test compound was determined at three different substrate concentrations, and was measured as the change in optical absorbance at 405 nm.
Test compound solutions (100 xcexcL; normally in buffer or saline containing BSA 10 g/L) were mixed with 200 xcexcL of human xcex1-thrombin (Sigma Chemical Co) in assay buffer (0.05 mol/L Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 xcexcL sample, together with 20 xcexcL of water, was added to 320 xcexcL of the substrate S-2238 (Chromogenix AB, Mxc3x6lndal, Sweden) in assay buffer, and the absorbance change (xcex94A/min) was monitored. The final concentrations of S-2238 were 16, 24 and 50 xcexcmol/L and of thrombin 0.125 NIH U/ml.
The steady state reaction rate was used to construct Dixon plots, i.e. diagrams of inhibitor concentration vs. 1/(xcex94A/min). For reversible, is competitive inhibitors, the data points for the different substrate concentrations typically form straight lines which intercept at x=xe2x88x92Ki.
Test D
Determination of Activated Partial Thromboplastin Time (APTT)
APTT was determined in pooled normal human citrated plasma with the reagent PTT Automated 5 manufactured by Stago. The inhibitors were added to the plasma (10 xcexcL inhibitor solution to 90 xcexcl plasma) and incubated with the APTT reagent for 3 minutes followed by the addition of 100 xcexcL of calcium chloride solution (0.025M) and APTT was determined in the mixture by use of the coagulation analyser KC10 (Amelung) according to the instructions of the reagent producer. The clotting time in seconds was plotted against the inhibitor concentration in plasma and the IC50APTT was determined by interpolation.
IC50APTT is defined as the concentration of inhibitor in human plasma that doubled the Activated Partial Thromboplastin Time.
Test E
Determination of Thrombin Time Ex Vivo
The inhibition of thrombin after oral or parenteral administration of the compounds of formula I and Ia, dissolved in ethanol:Solutol(trademark):water (5:5:90), were examined in conscious rats which, one or two days prior to the experiment, were equipped with a catheter for blood sampling from the carotid artery. On the experimental day blood samples were withdrawn at fixed times after the administration of the compound into plastic tubes containing 1 part sodium citrate solution (0.13 mol per L.) and 9 parts of blood. The tubes were centrifuged to obtain platelet poor plasma. The plasma was used for determination of thrombin time as described below.
The citrated rat plasma, 100 xcexcl, was diluted with a saline solution, 0.9%, 100 xcexcl, and plasma coagulation was started by the addition of human thrombin (T 6769, Sigma Chem Co, USA) in a buffer solution, pH 7.4, 100 xcexcl. The clotting time was measured in an automatic device (KC 10, Amelumg, Germany).
Where a compound of formula Ia was administered, concentrations of the appropriate active thrombin inhibitor of formula I in the rat plasma were estimated by the use of standard curves relating the thrombin time in the pooled citrated rat plasma to known concentrations of the corresponding xe2x80x9cactivexe2x80x9d thrombin inhibitor dissolved in saline.
Based on the estimated plasma concentrations of the active thrombin inhibitor of formula I (which assumes that thrombin time prolongation is caused by the aforementioned compound) in the rat, the area under the curve after oral and/or parenteral administration of the corresponding prodrug of formula Ia was calculated (AUCpd) using the trapezoidal rule and extrapolation of data to infinity.
The bioavailability of the active thrombin inhibitor of formula I after oral or parenteral administration of the prodrug of formula Ia was calculated as below:
[(AUCpd/dose)/(AUCactive,parenteral/dose]xc3x97100 
where AUCactive,parenteral represents the AUC obtained after parenteral administration of the corresponding active thrombin inhibitor of formula I to conscious rats as described above.
Test F
Determination of Thrombin Time in Urine Ex Vivo
The amount of the active thrombin inhibitor of formula I that was excreted in urine after oral or parenteral administration of the compounds of the invention, dissolved in ethanol:Solutol(trademark):water (5:5:90), was estimated by determination of the thrombin time in urine ex vivo (assuming that thrombin time prolongation is caused by the aforementioned compound).
Conscious rats were placed in metabolism cages, allowing separate collection of urine and faeces, for 24 hours following oral administration of compounds of the invention. The thrombin time was determined on the collected urine as described below.
Pooled normal citrated human plasma (100 xcexcL) was incubated with the concentrated rat urine, or saline dilutions thereof, for one minute. Plasma coagulation was then initiated by the administration of human thrombin (T 6769, Sigma Chem Company) in buffer solution (pH 7.4; 100 xcexcL). The clotting time was measured in an automatic device (KC 10; Amelung).
The concentrations of the active thrombin inhibitor of formula I in the rat urine were estimated by the use of standard curves relating the thrombin time in the pooled normal citrated human plasma to known concentrations of the aforementioned active thrombin inhibitor dissolved in concentrated rat urine (or saline dilutions thereof). By multiplying the total rat urine production over the 24 hour period with the estimated mean concentration of the aforementioned active inhibitor in the urine, the amount of the active inhibitor excreted in the urine (AMOUNTpd) could be calculated.
The bioavailability of the active thrombin inhibitor of formula I after oral or parenteral administration of the prodrug, was calculated as below:
[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose]xc3x97100
where AMOUNTactive,parenteral represents the amount excreted in the urine after parenteral administration of the corresponding active thrombin inhibitor of formula I to conscious rats as described above.
The invention is illustrated by way of the following examples. The amino acids Pro and Aze are defined as the S-isomers if not otherwise specified. The examples were obtained as diastereoisomers if not otherwise specified.